Method for analyzing chemical and or biological samples by means of particle images

ABSTRACT

A method for analyzing chemical and/or biological samples comprises the production of a particle image ( 42 ) of at least one particle included in the sample. Subsequently, a particle surface ( 10 ) of the at least one particle included in the particle image ( 42 ) is divided into particle zones ( 14,18 ). According to the invention, zone-dependent particle data are subsequently acquired in different states (z 1 , z 2 , z 3 ), which then can be evaluated.

This is a National Phase Application in the United States ofInternational Patent Application No. PCT/EP03/03960 filed Apr. 16, 2003,which claims priority on German Patent Application No. DE 102 16 683.8,filed Apr. 16, 2002.

The invention relates to a method for analyzing chemical and/orbiological samples by means of particle images, which is adapted to beperformed particularly in high- or medium-throughput screeninginstallations.

BACKGROUND OF THE INVENTION

For analyzing biological and/or chemical activities of substances orparticles and/or molecules present in the substances, data of thesamples to be analyzed are acquired. For acquiring data, microscopes,particularly confocal microscopes, are used. In doing so, certaincomponents of a sample are marked with fluorescent markers, where it ispossible to draw conclusions with respect to reactions within the sampleon the basis of the emission of fluorescence of these markers.

The reaction of a cell to a particular substance can be detected, forexample, by the migration of a molecule marked with a fluorescentmarker, for example, into the nucleus. An analyzing method suitabletherefor is described in U.S. Pat. No. 5,989,835. In this method, thenucleus of a cell is marked or colored in a first step. The coloring isperformed with a fluorescent marker that may be stimulated by a UVlaser. Further, a transcription factor in the cytoplasm surrounding thenucleus is marked with another fluorescent marker. By stimulating thenucleus with a UV laser and a threshold value method, a mask separatingthe nucleus from the cytoplasm is prepared. Subsequently, the mask isreduced in size so that it is guaranteed that a substantially circularfirst portion is exclusively arranged within the nucleus. In the nextstep, the mask border is enlarged so that a ring is created whichexclusively lies in the cytoplasm. By comparing the two cell portions,for example, it can be detected that an active substance marked with acolor marker has migrated into the nucleus, since the luminosity of thetwo portions changes in this case.

First, this method described in U.S. Pat. No. 5,989,835 has thedisadvantage that the nucleus has to be located. This is only possibleby using special colors or special color markers since it has to beensured that they react with the nucleus to be able to detect theposition of the nucleus. The color markers used in this case, Hoechst33342 and 33258, for example, can only be stimulated by a UV laser. Theacquisition of UV lasers, however, is expensive, and they have highoperational costs due to their high cooling water consumption. Anotherdisadvantage of the afore-described method consists in that meaningfulresults can only be achieved if the ring is arranged completely withinthe cytoplasm. This is not the case, for example, when the nucleus isarranged in the border portion of the cytoplasm. Another disadvantage ofthe afore-described method consists in that always a definitely definedsurface in the form of the nucleus is defined and detected. Thus, themethod is expensive and inflexible.

It is the object of the invention to provide an analyzing method forchemical and/or biological samples where the trouble with detectingchanges of the sample is reduced.

This object is solved, according to the invention, by the method foranalyzing chemical and/or biological samples, particularly withhigh-throughput or medium-throughput screening installations, with thesteps of: respectively producing at least one particle image (42) of atleast one sample with at least one particle being included in thesample, respectively; (b) defining several particle zones (14,18; 22,24;32,34,36) independent of subparticular compartments; (c) acquiringparticle data of the sample independently of the zone; and (d)evaluating the acquired particle data.

SUMMARY OF THE INVENTION

According to the method according to the invention, which isparticularly suitable for high- and medium-throughput screeninginstallations, particle images of at least one sample are produced in afirst step. These images include at least one particle, e.g., a cell,included in the sample. Typically, each image includes more than 10,particularly more than 50 particles, particularly cells. Producing theparticle image is effected, for example, by means of a microscope and/oran image acquisition means such as a CCD camera that may be connectedwith a suitable image evaluating means.

According to the invention, particle zones are defined which areindependent of subparticular compartments. In this connection, the zonesare preferably arranged or defined within a particle image.

Preferably, a particle surface, i.e., the surface occupied by a particleincluded in the produced image, is divided into several particle zones.In this case, the particle surface is defined by the fact that theforeground differs from the background in the particle image. To thisend, a threshold value can be preset which defines the boundary betweenbackground and foreground. In dependence on the selected thresholdvalue, the particle surface defined in the particle image comprises theactually present surface of the particle or possibly a surface slightlysmaller or larger. Preferably, the particle surface is plane. In thiscase, it is advantageous to use confocal microscopes since definiteplanes within the sample can be defined by confocal microscopes so thata definite particle surface can be defined in the particle image.

Each particle surface of the particle images which are preferablyproduced independently of each other of at least one sample are dividedinto particle zones according to the invention. The division of theparticle zones is effected independently of subparticular compartments.If the particles are cells, for example, the division of the particlesurface into particle zones is effected independently of subcellularcompartments such as, for example, nuclei, mitochondria, lysosomes etc.Preferably, the particle surface is divided into at least five,particularly preferably into at least ten particle zones.

According to the invention, for example, several zone images of a singlesample can be produced at different points of time. Then, these zoneimages can be, for example, compared with each other for evaluation. Itis as well possible to produce particle images of several samples at thesame points of time and to compare them with each other. In this case,for example, one sample may serve as reference sample to which noreagent has been added. A combination of these two procedures is alsopossible.

It is also possible that several particle images are acquired and atleast one particle zone is defined that extends over several particleimages. This is a particle zone that is not arranged within a preferablyplane particle surface but in space. Such a particle zone may have aplane or spatial configuration. In case of a spatial configuration,defined geometric shapes such as cuboids, cubes or the like arepreferred.

In the next step, particle data of the sample or possibly of differentsamples and the particle images, respectively, are acquired independence on the zones. Thus, for example, an acquisition of the mediumluminosity of individual zones with different samples is effected. Theindividually produced particle images of several samples show theparticles in different states since, for example, different reagentsadded to the samples cause different reactions in the samples. In onesample, for example, a reagent marked with a marker will penetrate intothe cell and in another sample, it will stay outside the cell plasm. Inthis case, the acquired luminosities of the individual zones of thedifferent particle images are different, for example, whereas theacquired luminosities of the individual zones were substantiallyidentical for all samples before the addition of the different reagents.The particle data acquired in this connection are then evaluated in thenext step. Because of a luminosity shift within the particle zones, forexample, conclusions can be drawn with respect to the movement of activesubstances marked with a color marker or the like. Instead of usingcolor markers, the characteristic radiation of suitable particles can bedetected as well. Here, the fluorescence of color markers as well as thecharacteristic radiation may be in the visible as well as in theinvisible range.

According to the method according to the invention, it is thus notrequired to add a special color for marking the nucleus or the like tothe sample since the position of the nucleus, for example, does not haveto be known for carrying out the method according to the invention.Other subparticular compartments do not have to be determined in detaileither for carrying out the method according to the invention. Theparticle surface is rather divided into particle zones independent ofsubparticular compartments. Thus, it is not required to usecorresponding colors that can only be stimulated by a UV laser expensivein acquisition and operation. Thus, the costs of the analyzing methodcan be considerably reduced. Since, for example, the definition of theindividual particle and cell zones, respectively, is independent of theposition of the nucleus in the analysis of cells, the described problemsoccurring with the position of the nucleus at the border of thecytoplasm in the method described in U.S. Pat. No. 5,989,835 cannotoccur with the method according to the invention. Thus, the methodaccording to the invention is considerably more flexible.

Preferably, a definition of the particle zones is respectively effectedbefore the acquisition and subsequent evaluation of the particle data.

Preferably, several samples including a plurality of particles each areanalyzed simultaneously in the method according to the invention. Indoing so, the produced particle images of the samples are compared. Atthe same point of time, the individual samples are in respectivelydifferent states. By comparing the particle images, i.e., the individualzones of the particle images, a migration of a reagent provided with acolor marker, for example, or other changes within the particle can beperceived.

Further, it is possible to define particle zones such that they lieoutside the particle. By the same comparisons of the particle zones,movements of color markers outside the particles can be perceived, forexample.

Preferably, the sample is colored before the particle image will beproduced. By these colorations which can be done with conventionalcolors such as fluorescent markers, the cells or the liquid surroundingthe cells is colored, for example. By this coloration, the boundary ofthe cells and the particles, respectively, can be detected. It is notrequired to use special colors for coloring the nuclei. Since theparticle boundaries can be detected better by coloring the sample, theparticle surface of the individual particles can be determined moreeasily by an image processing system, confocal microscopes or otherconfocal optics means being preferably used for determining the particlesurfaces to be able to acquire particularly individual particles,particularly cells, existing in a particular plane of the sample.Particularly upon coloring the sample, it is essential that theforeground of the sample can be distinguished from the background of thesample by the image processing system, particularly the confocalmicroscope. The exact particle boundary does not have to be determined.It is rather sufficient when a particle image can be produced wherein atleast a large part of the particle is divided into particle zones.

Preferably, the definition of the particle zones is effected such thatthe entire particle surface is divided into particle zones. Theindividual particle zones are thus immediately adjacent to each otherwithout any clearance. This has the advantage that movements of, forexample, active substances marked with a color marker can be observedclosely. It is thus possible to detect movement directions of individualor several active substances or the like. Further, it is possible todefine zones outside the particle surface as well. Thereby, it can bedetected by a luminosity comparison, for example, how many of theparticles marked with a color marker are respectively located within andwithout a particle, such as a cell. The border portions of the particlezones may also comprise portions directly next to the particle so thatthese are mixed zones. According to the invention, however, the particleboundary is preferably determined by coloring the sample, for example,so that either only particle zones are defined or particle zones andouter zones or only outer zones can be defined.

When evaluating the particle data, it is possible, according to theinvention, not to acquire the particle data of all zones but to acquirethe particle data of selected zones only. It is possible, for example,to observe only a border zone extending along the boundary line of theparticle and to evaluate its particle data only. It is also possible toobserve only the differences of the luminosities of a single zone perparticle image.

Preferably, the definition of the particle zones is effected accordingto a mathematical model. Preferably, in this connection, mathematicalmodels are used which are suited to the reactions to be expected. Thestarting point is a particle image, respectively.

Further, upon defining the particle zones, it is possible to define themin such a manner that all zones have the same surface area. This isparticularly advantageous upon comparing the medium luminosities ofindividual zones with each other since the luminosity change cannot becaused or influenced by a dimensional change of the zone.

The method according to the invention is particularly suitable foranalyzing samples including cells. Preferably, a particle included inthe sample is a cell which may form part of a cell compound.Particularly, the particle exclusively consists of one or more cells.With respect to the preferred analysis of cells, the core of theinvention therefore consists in that several cell zones independent ofsubcellular compartments are preferably defined in a particle surface,i.e., a cell surface.

Preferably, the particle state, particularly the cell state, is analyzedin dependence on substances added to the sample. In this connection, themethod according to the invention is particularly well suited to drugscreening. Samples of different cell states are analyzed, for example,the cell state particularly comprising apoptosis, necrosis,translocation of cellular components, internalization of membraneousmolecules or molecule complexes, cell differentiation, morphologicalappearance, splitting of subcellular compartments and/or generation ofsubcellular compartments.

BRIEF DESCRIPTION OF THE DRAWINGS

Hereinafter, the invention will be explained in detail with respect topreferred embodiments with reference to the accompanying drawings. Inthe Figures:

FIGS. 1-3 show sketches of particle zones defined by different methods,

FIG. 4 shows particle images of an analyzed example in different states,

FIG. 5 shows a diagram of the medium luminosity signals with referenceto the individual zones of the particle images illustrated in FIG. 4,

FIG. 6 shows particle images of a further analyzed example in differentstates, and

FIG. 7 shows a diagram of the surface over the zones of the particleimages illustrated in FIG. 6.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, three different methods according to the invention, fordividing particle surfaces into particle zones, are explained withrespect to FIGS. 1-3.

In FIG. 1, a particle surface 10 is illustrated, the outer line 12representing the boundary line of the particle, such as the cell. Theboundary line need not necessarily be the exact boundary of theparticle. According to the invention, it is rather sufficient to definea boundary between the background and the foreground preset independence on a predetermined threshold value as a boundary line. Inthis case, the division of the particle surface into particle zones iseffected by the fact that each zone has an inner and an outer boundaryline. An outer zone 14, for example, has the boundary line 12 as anouter boundary line and the boundary line 16 as an inner boundary line.The next zone 18 farther inward has the boundary line 16 as an outerboundary line and the boundary line 20 as an inner boundary line and soforth. The definition of the boundary lines 16,20 is effected by thefact that each of them has a substantially constant distance to theparticle boundary 12.

Thus, the individual zones 14,18 can also be described by the followingformula, for example, which defines the interval of the distance to theparticle boundary:(N*Δd,N*Δd+Δd)where N=0, 1, . . . andΔd is a width of the zone.

By the above formula, the distance of individual strip-shaped zones14,18 to the particle boundary 12 is thus defined in dependence on Δd.In this connection, it is decisive that all zones 14,18,20 have the samewidth.

With this method, it is further possible to also define zones arrangedoutside the particle surface 10.

FIG. 2 shows another method for determining particle zones 22,24. Theparticle zones 22,24 defined here are also divided from each other byboundary lines 26,28,30. The zones 22,24, however, do not have the samewidth. For determining these zones, concentric boundary lines 26,28,30are rather used. Thus, the boundary lines 26,28,30 have similargeometric shapes. Stated in simplified terms, the boundary line 28 maybe produced by a percental reduction of the size of the boundary line26.

For defining the zones of one of the two methods apparent in FIGS. 1 and2, the definition of boundary lines is not absolutely necessary. Themethod may also be performed by dividing the particle image intoindividual pixels and defining the affiliation of a pixel to the one orthe other zone by mathematical formulas. This results in boundary linesbetween the adjacent particle zones, of course.

Different particle zones 32,34,36 etc. can also be defined with a methodapparent from FIG. 3. Here, a main point 38 is defined, which is, forexample, the most luminous point within the particle surface 10, thegeometric center or the center of gravity. Starting from this point,preferably radially extending boundary lines 40 serving to delimit theparticle zones 32,34,36 etc. are defined. Preferably, the aperture angleof the individual boundary lines 40 is constant. The individual boundarylines may also be arranged such that the surface areas of the zones32,34,35 are constant.

In the first example for explaining the method according to theinvention represented with respect to FIGS. 4 and 5, three particleimages 42 are illustrated in FIG. 4, which show different states z1, z2,and z3 of various particle images 44 from different samples. Further,the zone images 44 to the corresponding states are illustrated, in whichzone images the cells 46 have been divided into zones by means of themethod described in detail with respect to FIG. 1.

In the illustrated example, different reagents are introduced intoseveral identical samples. After a fixed time interval of possiblyseveral hours, the samples of which there are three in the illustratedexample are observed and a particle image 46 as well as a zone image 44is prepared of each of the three samples. Thus, three different zoneimages 44 are prepared which represent different states z1, z2, and z3in the three samples. In the first sample that is on the left in FIG. 4,there was no reaction to the substrate added to the sample. Independence on the individually defined zones, this results in the courseillustrated in FIG. 5 with respect to the state z1. In the second sampleillustrated in the middle of FIG. 4, the substrate has caused areaction. Here, the substrate has migrated into the interior of thecell. It is apparent from the diagram in FIG. 5 that this has resultedin an increase in luminosity in the region of the numerals 20-23 whichrepresent corresponding zones. A corresponding luminosity increase isalso apparent in the third sample on the right in FIG. 4. This, in turn,is particularly apparent from the diagram illustrated in FIG. 5 in theregion of the numerals 17-20.

From the example illustrated in FIGS. 6 and 7, it can be seen that thegrowth of axons in nerve cells can be simply detected by means of themethod according to the invention.

In FIG. 6, three particle images 44 are illustrated in different statesz1, z2, and z3. The cell, in turn, is divided into individual particlezones by means of the zone definition method described in FIG. 1, saidparticle zones not being illustrated in detail here. In the illustratedexample, in turn, different substrates have been added to three samples.After a preset time interval that may, in turn, possibly last severalhours, the three samples are analyzed. A particle image as well as aparticle image 44 are produced of each of the samples. Then, the threeparticle images 44 show different states z1, z2, and z3 of differentcells, i.e., states in the individual samples. It is already apparentfrom the representations in FIG. 6 that particularly the cells in thethird sample (right image in FIG. 6) react to the added substrate andform long axons. In the middle sample, an axon was formed as well,although it was not so long. In the left sample, it is apparent that noreaction has occurred. Depending on the length of the formed axon, thesize of the surface of the outer zone increases differently in differentsamples. This is particularly apparent from the diagram illustrated inFIG. 7. The surface of the outer zone illustrated on the left in FIG. 7is larger in the state z3 than in the state z1. Thus, it is onlypossible to detect in which cells axons have grown and in which cellsthey have not grown by comparing the curves illustrated in FIG. 7.

1. Method for analyzing chemical and/or biological samples withhigh-throughput or medium-throughput screening installations, comprisingthe steps of: producing one or more particle images of at least onesample with at least one particle, having a particle surface, beingincluded in the sample; defining several particle zones of the particlesurface by determining a particle boundary before the particle zones aredefined and defining a main point within the particle surface andboundary lines extending radially therefrom to the particle boundary,wherein the several particle zones of the particle surface are definedindependent of subparticular compartments; acquiring particle data ofthe sample in dependence on the particle zones; and evaluating theacquired particle data.
 2. Method according to claim 1, wherein aplurality of particle images are produced and at least one particle zoneis defined that extends over several particle images of the plurality ofparticle images.
 3. Method according to claim 1, wherein severalparticle images are produced of several samples.
 4. Method according toclaim 1, wherein the particle zones are defined within the particlesurface of the at least one particle included in the particle image. 5.Method according to claim 1, wherein substantially the entire particlesurface is divided into particle zones.
 6. Method according to claim 1,wherein the particle zones are defined immediately before theacquisition of the particle data.
 7. Method according to claim 1,wherein several particle images are produced of the at least one sampleat different points of time.
 8. Method according to claim 7, whereindifferent states of the at least one particle included in the sample arecompared with each other when evaluating the acquired particle data. 9.Method according to claim 1, wherein samples of different particlestates are analyzed at different times.
 10. Method according to claim 1,wherein the sample is colored before the particle image is produced. 11.Method according to claim 1, wherein the boundary lines extendingradially from the main point to the particle boundary define first,second and third particle zones.
 12. Method according to claim 1,wherein the most luminous point within the particle surface is definedas the main point.
 13. Method according to claim 1, wherein the radiallyextending boundary lines have a substantially identical aperture anglewith respect to each other.
 14. Method according to claim 1, whereineach individual particle zone of the several particle zones is definedso that each individual particle zone has substantially the same surfacearea as each of the other individual particle zones.
 15. Methodaccording to claim 1, wherein individual particle zones of the severalparticle zones are selected for analysis in dependence on selectioncriteria.
 16. Method according to claim 8, wherein particle dataacquired in dependence on the particle zones includes size of theparticle zones in different states of the at least one particle. 17.Method according to claim 8, wherein particle data acquired independence on the particle zones includes luminosity of the particlezones in different states of the at least one particle.
 18. Methodaccording to claim 1, wherein the at least one particle is a cell andparticle zones, independent of subcellular compartments, are defined.19. Method according to claim 18, wherein the cell forms part of a cellcompound comprising one or more cells.
 20. Method according to claim 18,wherein samples of different cell states are analyzed and the cell statecomprises one or more of components selected from the group consistingof apoptosis, necrosis, translocation of cellular components,internalization of membranous molecules or molecule complexes, celldifferentiation, morphological appearance, splitting of subcellularcompartments and generation of cellular compartments.
 21. Methodaccording to claim 20, wherein the cell state is influenced by addingchemical substances, biological substances, or chemical and biologicalsubstances.
 22. Method according to claim 21, wherein the cell state isinfluenced by adding potential pharmacological active substances. 23.Method according to claim 1, wherein the particle surface is dividedinto at least five particle zones.
 24. Method according to claim 23,wherein the particle surface is divided into at least ten particlezones.
 25. Method according to claim 1, further comprising the steps of:presetting a threshold value for defining a boundary between backgroundand foreground in the one or more particle images; and defining theparticle surface by a difference between the background and theforeground that exceeds the threshold value.